The invention relates to compositions and methods for use in treating skeletal system disorders in a vertebrate at risk for bone loss, and in treating conditions that are characterized by the need for bone growth, in treating fractures, and in treating cartilage disorders. More specifically, the invention concerns the use of inhibitors of proteasomal activity and of NF-xcexaB activity for this purpose.
Bone is subject to constant breakdown and resynthesis in a complex process mediated by osteoblasts, which produce new bone, and osteoclasts, which destroy bone. The activities of these cells are regulated by a large number of cytokines and growth factors, many of which have now been identified and cloned.
There is a plethora of conditions which are characterized by the need to enhance bone formation or to inhibit bone resorption. Perhaps the most obvious is the case of bone fractures, where it would be desirable to stimulate bone growth and to hasten and complete bone repair. Agents that enhance bone formation would also be useful in facial reconstruction procedures. Other bone deficit conditions include bone segmental defects, periodontal disease, metastatic bone disease, osteolytic bone disease and conditions where connective tissue repair would be beneficial, such as healing or regeneration of cartilage defects or injury. Also of great significance is the chronic condition of osteoporosis, including age-related osteoporosis and osteoporosis associated with post-menopausal hormone status. Other conditions characterized by the need for bone growth include primary and secondary hyperparathyroidism, disuse osteoporosis, diabetes-related osteoporosis, and glucocorticoid-related osteoporosis.
There are currently no satisfactory pharmaceutical approaches to managing any of these conditions. Bone fractures are still treated exclusively using casts, braces, anchoring devices and other strictly mechanical means. Further bone deterioration associated with post-menopausal osteoporosis has been treated with estrogens or bisphosphonates, which may have drawbacks for some individuals. Although various approaches have been tried, as further discussed below, there remains a need for additions to the repertoire of agents which can be used to treat these conditions.
Treatment of bone or other skeletal disorders, such as those associated with cartilage, can be achieved either by enhancing bone formation or inhibiting bone resorption or both. A number of approaches have been suggested which relate to bone formation.
Bone tissue is an excellent source for factors which have the capacity for stimulating bone cells. Thus, extracts of bovine bone tissue obtained from slaughterhouses contain not only structural proteins which are responsible for maintaining the structural integrity of bone, but also biologically active bone growth factors which can stimulate bone cells to proliferate. Among these latter factors are transforming growth factor xcex2, the heparin-binding growth factors (e.g., acidic and basic fibroblast growth factor), the insulin-like growth factors (e.g., insulin-like growth factor I and insulin-like growth factor II), and a recently described family of proteins called bone morphogenetic proteins (BMPs). All of these growth factors have effects on other types of cells, as well as on bone cells.
The BMPs are novel factors in the extended transforming growth factor xcex2 superfamily. Recombinant BMP2 and BMP4 can induce new bone formation when they are injected locally into the subcutaneous tissues of rats (Wozney J Molec Reprod Dev (1992) 32:160-67). These factors are expressed by normal osteoblasts as they differentiate, and have been shown to stimulate osteoblast differentiation and bone nodule formation in vitro as well as bone formation in vivo (Harris S. et al. J Bone Miner Res (1994) 9:855-63). This latter property suggests potential usefulness as therapeutic agents in diseases which result in bone loss.
The cells which are responsible for forming bone are osteoblasts. As osteoblasts differentiate from precursors to mature bone-forming cells, they express and secrete a number of enzymes and structural proteins of the bone matrix, including Type-1 collagen, osteocalcin, osteopontin and alkaline phosphatase. They also synthesize a number of growth regulatory peptides which are stored in the bone matrix, and are presumably responsible for normal bone formation. These growth regulatory peptides include the BMPs (Harris S. et al. (1994), supra). In studies of primary cultures of fetal rat calvarial osteoblasts, BMPs 1, 2, 3, 4, and 6 are expressed by cultured cells prior to the formation of mineralized bone nodules (Harris S. et al. (1994), supra). Like alkaline phosphatase, osteocalcin and osteopontin, the BMPs are expressed by cultured osteoblasts as they proliferate and differentiate.
Although the BMPs are potent stimulators of bone formation in vitro and in vivo, there are disadvantages to their use as therapeutic agents to enhance bone healing. Receptors for the bone morphogenetic proteins have been identified in many tissues, and the BMPs themselves are expressed in a large variety of tissues in specific temporal and spatial patterns. This suggests that BMPs may have effects on many tissues in addition to bone, potentially limiting their usefulness as therapeutic agents when administered systemically. Moreover, since they are peptides, they would have to be administered by injection. These disadvantages impose severe limitations to the development of BMPs as therapeutic agents.
The fluorides, suggested also for this purpose, have a mode of action which may be related to tyrosine phosphorylation of growth factor receptors on osteoblasts, as described, for example, Burgener et al. J Bone Min Res (1995) 10:164-171, but administration of fluorides is associated with increased bone fragility, presumably due to effects on bone mineralization.
Small molecules which are able to stimulate bone formation have been disclosed in PCT applications WO98/17267 published Apr. 30, 1998, WO97/15308 published May 1, 1997 and WO97/48694 published Dec. 24, 1997. These agents generally comprise two aromatic systems spatially separated by a linker. In addition, PCT application WO98/25460 published Jun. 18, 1998 discloses the use of the class of compounds known as statins in enhancing bone formation. U.S. application Ser. No. 09/096,631 filed Jun. 12, 1998 is directed to compounds for stimulating bone growth that are generally isoprenoid pathway inhibitors. The contents of this application, as well as of the PCT applications cited above, are incorporated herein by reference.
Other agents appear to operate by preventing the resorption of bone. Thus, U.S. Pat. No. 5,280,040 discloses compounds described as useful in the treatment of osteoporosis. These compounds putatively achieve this result by preventing bone resorption.
Wang, G.-J. et al., J Formos Med Assoc (1995) 94:589-592 report that certain lipid clearing agents, exemplified by lovastatin and bezafibrate, were able to inhibit the bone resorption resulting from steroid administration in rabbits. There was no effect on bone formation by these two compounds in the absence of steroid treatment. The mechanism of the inhibition in bone resorption observed in the presence of steroids (and the mechanism of the effect of steroid on bone per se) is said to be unknown.
An abstract entitled xe2x80x9cLovastatin Prevents Steroid-Induced Adipogenesis and Osteoporosisxe2x80x9d by Cui, Q. et al. appeared in the Reports of the ASBMR 18th Annual Meeting (September 1996) J Bone Mineral Res. (1996) 11(S1):S510 which reports that lovastatin diminished triglyceride vesicles that accumulated when osteoprogenitor cells cloned from bone marrow stroma of chickens were treated in culture with dexamethasone. Lovastatin was reported to diminish the expression of certain mRNAs and to allow the cells to maintain the osteogenic phenotype after dexamethasone treatment, and chickens that had undergone bone loss in the femoral head as a result of dexamethasone treatment were improved by treatment with lovastatin.
These data are, however, contrary to reports that dexamethasone and other inducers, such as BMPs, induce osteoblastic differentiation and stimulate osteocalcin mRNA (Bellows, C. G., et al., Develop Biol (1990) 140:132-38; Rickard, D. J., et al., Develop Biol (1994) 161:218-28). In addition, Ducy, P. et al., Nature (1996) 382:448-52 have recently reported that osteocalcin deficient mice exhibit a phenotype marked by increased bone formation and bones of improved functional quality, without impairment of bone resorption. Ducy et al. state that their data suggest that osteocalcin antagonists may be of therapeutic use in conjunction with estrogen replacement therapy (for prevention or treatment of osteoporosis).
It has also been shown that lovastatin inhibits lipopolysaccharide-induced NF-xcexaB activation in human mesangial cells. Guijaro, C. et al. Nephrol Dial Transplant (1996) 11:6:990-996.
The present invention discloses convenient assays for compounds that will be useful in the treatment of bone disorders. The assay involves inhibition of the activity of the transcription factor NF-xcexaB or the activity of proteasomal proteases. Compounds which inhibit these activities are generally useful in treating bone disorders. Their ability to do so can be further confirmed by additional assays.
It has recently been shown that mice lacking expression of the transcription factor NF-xcexaB develop an abnormal bone condition, osteopetrosis (the converse of osteoporosis), due to an absence of osteoclast formation (Franzoso, G. et al. Genes and Dev (1997) 11:3482-3496; Iotsova, V. et al. Nature Med (1997) 3:1285-1289). Osteopetrosis is characterized by such an absence of osteoclast function and the filling in of the marrow cavity with osteocartilagenous material. The mice showed no abnormal osteoblast function. It is therefore surprising that compounds that inhibit NF-xcexaB activity are able to directly stimulate bone formation. Similarly, the ability of proteasome inhibitors to stimulate bone growth is unexpected in light of these results, since proteasome inhibitors are expected to function as NF-xcexaB inhibitors as well. This is because NF-xcexaB must enter the nucleus to exert its effects on specific target genes, and compounds that inhibit its entry into the nucleus effectively inhibit its activity. Proteasome activity is required for NF-xcexaB translocation. NF-xcexaB is present in the cytoplasm bound to the inhibitory proteins IxcexaBxcex1 and IxcexaBxcex2 which prevent its translocation. Translocation occurs when kinases phosphorylate IxcexaBxcex2 to cause its degradation by proteasome activity, thus resulting in its release for entry into the nucleus. Inhibition of proteasome activity prevents this release and thus effectively inhibits NF-xcexaB.
The proteasome is a noncompartmentalized collection of unrelated proteases which form a common architecture in which proteolytic subunits are self-assembled to form barrel-shaped complexes (for review, see Baumeister et al., Cell (1998) 92:367-380. The proteasome contains an array of distinct proteolytic activities inside eukaryotic cells. As set forth above, compounds which inhibit proteasomal activity reduce NF-xcexaB activity by limiting its capacity to be translocated to the nucleus (Barnes, P. J. et al. New Engl J Med (1997) 336:1066-1071.
The present invention adds to the repertoire of osteogenic agents by providing potential drugs which would inhibit key proteins and enzymes involved in proteasomal activity and which decrease the activity of the nuclear transcription factor NF-xcexaB, and thus stimulate bone growth. In accordance with the present invention, we have discovered that inhibition of the functions of the proteasomal proteins and the transcription factor NF-xcexaB in bone cells leads to increased bone formation. Thus, assessing a candidate compound for its ability to inhibit proteasomal proteins or NF-xcexaB provides a useful means to identify bone growth anabolic agents.
The present specification provides methods for identification of osteogenic compounds to stimulate bone growth by assessing their capacity to inhibit proteasome activity or to inhibit the activity of the transcription factor NF-xcexaB in bone cells. Also useful in the methods of the invention are compounds which inhibit the in situ production of the enzymes contained in the proteasome or inhibit the production of NF-xcexaB. Once a compound found to inhibit these activities has been identified, it can be used in an additional aspect of the inventionxe2x80x94a method to stimulate the growth of bone by contacting suitable cells with the identified compound. The cellular contact may include in vivo administration and the compounds of the invention are thus useful in treating degenerative bone diseases, fractures, dental problems and the like. These methods are performed, according to the present invention, with compounds identified as inhibitors of proteasome activity, inhibitors of the activity of transcription factor NF-xcexaB, or inhibitors of the production of the proteasome enzymes or NF-xcexaB, other than compounds which have these activities but also inhibit the isoprenoid pathway.
It has, surprisingly, also been found that agents that inhibit NF-xcexaB and/or proteasomal activity or inhibit the production of these proteins, also stimulate hair growth. Thus, the method described above can also be used to identify compounds that are useful in hair growth stimulation. In a further embodiment, the present invention provides a method of regulating the hair follicle life cycle and promoting hair growth. This method comprises contacting hair follicles with the identified compound so as to effect the desired growth.